CN113980813A

CN113980813A - Breeding method of high-temperature-resistant yarrowia lipolytica variant strain and application of breeding method in erythritol fermentation

Info

Publication number: CN113980813A
Application number: CN202111214391.3A
Authority: CN
Inventors: 裴疆森; 李红娇; 董建辉; 郝飞克; 栾春光; 金玮鋆; 于佳俊
Original assignee: China National Research Institute of Food and Fermentation Industries
Current assignee: China National Research Institute of Food and Fermentation Industries
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2022-01-28
Anticipated expiration: 2041-10-19
Also published as: CN113980813B

Abstract

The invention relates to a method for directionally breeding yarrowia lipolyticaYarrowia lipolytica) The breeding method of the high-temperature resistant variant strain can obviously improve the ultimate tolerance temperature of the strain, and the method is used for transforming the erythritol fermentation strain to obtain the yarrowia lipolytica GDMCC 61939, so that the optimal fermentation temperature of the strain is improved to be more than 34 ℃ from 29 ℃ of the original strain, thereby reducing the cooling load in the fermentation process, and having obvious application valueThe value is obtained.

Description

Breeding method of high-temperature-resistant yarrowia lipolytica variant strain and application of breeding method in erythritol fermentation

Technical Field

The invention relates to the modification and application of industrial microorganisms, and belongs to the technical field of bioengineering.

Background

Yarrowia lipolytica (Yarrowia lipolytica) Is a typical mesophilic aerobic yeast strain. Because of the characteristics of safety, easy culture, carbon source utilization and the like, the method is widely used for producing products such as lipase, organic acid and the like at present and is widely used as a host bacterium of genetic engineering for expressing exogenous genes. However, the growth temperature limit of yarrowia lipolytica is very strict, and the limit tolerance temperature of almost all wild strains can only reach 32 ℃, so that the application of the yarrowia lipolytica in some occasions is greatly limited, particularly in the occasions of high cell density and large volume fermentation, and growth retardation, product yield reduction and even yeast cell death are caused by large-scale heat release and temperature rise of a system. With the increasing global environment temperature and the tightening carbon emission requirement, the temperature reduction cost required by the temperature control of the fermentation system will increase day by day, so the improvement of the temperature tolerance of the yarrowia lipolytica strain has urgent practicability.

In recent years, numerous studies have shown that high calorie sugar intake can bring adverse effects to health, and consumption of low calorie sweeteners is an important way to meet people's demand for sweetness and is generally accepted by food and beverage manufacturers and by consumers in general. Among the low-calorie sweeteners, sugar alcohol compounds, among which erythritol is the most prominent representative of so-called bulk, zero-calorie sweeteners, are highly valued in the industry for their good sweetness and biological tolerance, and have good market capacity and development prospects. At present, yarrowia lipolytica in strains capable of being used for erythritol production is the only strain which is internationally recognized as safe (GRAS), the research and the modification of the erythritol fermentation process of the strain are generally regarded as important, and the research of improving the high-temperature tolerance of erythritol is an important field of the research of the biotechnology.

At present, the method for obtaining the yarrowia lipolytica high-temperature tolerant strain mainly comprises two methods, namely a yeast strain adversity domestication breeding method and a gene engineering method. The former is a method of selecting variant strain with high temperature resistant apparent character from the strain by continuously and repeatedly increasing the culture temperature of the strain for a long time, the selection method has long screening period, and the obtained high temperature resistant character has poor stability and fast strain performance degradation, and the defects of the polyploid fungi such as saccharomycetes are more prominent. The latter is to integrate the high temperature resistant gene obtained from exogenous gene donor organism, such as the gene coding heat shock protein HSP, into the genome of yarrowia lipolytica, so that it has certain high temperature resistance. However, the conversion power of the method is low, the influence on the production characteristics of strains can be large, and global cell biology optimization is also needed to obtain good strains for industrial application. In addition, the application of the engineering bacteria obtained by using a genetic engineering method in food and ingredients thereof is limited by great regulations and ethics in many countries and regions at present. There is therefore a need for other efficient methods to obtain a more temperature tolerant approach.

Previous studies have shown that the heat resistance of microorganisms depends greatly on the damage of cell structures caused by oxidative stress (ROS) pressure, and ROS in yeast cells under aerobic conditions are mainly generated by the oxidative respiratory chain of mitochondrial organelles, so that the reduction of the reaction strength of the respiratory chain can effectively reduce the generation of the ROS, thereby reducing the damage to active elements such as cell membrane systems and the like. Most proteins that function in subcellular elements such as mitochondria are encoded by the chromosomal genome, synthesized in the cytoplasm and transported to organelles, and signal peptides that determine the localization of proteins play an important role in the localization of isozymes in cells. And the signal peptide sequence of yarrowia lipolytica contains a high proportion of basic amino acids, such as arginine and lysine residues, so that the oxidation activity of mitochondria is expected to be reduced by reducing the synthesis and supply of the basic amino acids of yeast cells, thereby improving the temperature adaptability of strains.

The invention screens basic amino acid defect marks from a yeast mutation phenotype library obtained by physical and chemical mutagenesis, efficiently screens out stable mutant strains with high temperature resistance, and applies the method to the breeding of erythritol-producing yeast strains to obtain strains capable of fermenting at high temperature.

Disclosure of Invention

The invention aims to provide a method suitable for screening high-temperature-resistant characters of yarrowia lipolytica, and a new strain suitable for erythritol fermentation is bred by the method, so that the temperature tolerance of the strain is improved, the cooling load in the fermentation process is reduced, and the production cost is reduced.

Strain preservation information: the strain name is as follows: yarrowia lipolytica R27, accession number: 61939, date of deposit: 2021, 9/16, storage site: guangdong province microbial strain preservation center, No. 59 building 5 of Jie No. 100 Dazhong, Jieli, Guangzhou city.

In order to achieve the purpose, the invention adopts the following technical scheme.

The invention relates to a yarrowia lipolytica strain transformation technology, which takes common wild yarrowia lipolytica CGMCC No.1431 as an original strain and causes genetic variation through physicochemical and molecular biological methods, wherein various phenotypic variations are involved. From these, arginine auxotrophic strains are selected, from which variants having stable high temperature tolerance can be suspended.

Further, the operation is carried out according to the following steps:

(1) and processing the yarrowia lipolytica cells by using chemical and physical mutagenesis methods such as EMS, UV and the like and other molecular mutagenesis methods to obtain a yeast variation library.

(2) Screening arginine auxotrophic variants that grew only on complete medium at room temperature and not on synthetic medium containing arginine-free YNB as a nitrogen source.

(3) Verifying the high temperature tolerance difference between the control original strain and the arginine auxotrophic strain, and selecting the high temperature resistant variant strain from the comparison original strain and the arginine auxotrophic strain.

The method for producing erythritol by using the yarrowia lipolytica of the invention is characterized by comprising the steps of screening high-temperature-resistant arginine auxotrophic strains and then carrying out erythritol fermentation by using glucose.

Further, the method comprises the following specific steps:

(1) strain activation

The mutants were streaked on YNB solid medium plate containing arginine, and cultured at 34. + -. 1 ℃ for 72 hours.

(2) Seed culture

And (3) under an aseptic condition, selecting a single colony on the plate obtained in the step (1), inoculating a fermentation culture medium, and carrying out shake culture at 34 +/-1 ℃ for 24 hours to obtain a seed solution.

(3) Fermentation culture

Inoculating the seed liquid into a fermentation culture medium in an inoculation amount of 0.1-20%, and performing shake culture at 30 +/-1 ℃ for 12 +/-2 hours to obtain a fermentation liquid.

Wherein, the formula of the culture medium in the step (1) is as follows: 100mg of arginine, 100g of glucose and 20g of agar were added to 1L of water, and sterilized at 115 ℃ for 30 minutes. When the medium was not cooled, 10 XYNB solution without amino acids was filtered through a sterile millipore filter into the medium to give a final addition of 6.7 g.

The seed culture medium formula in the step (2) is as follows: 3g of yeast extract powder, 5g of peptone and 100g of glucose are added into 1L of water, and the mixture is sterilized at 115 ℃ for 30 minutes.

The formula of the fermentation medium in the step (3) is as follows: 3g of yeast extract powder, 5g of peptone and 300g of glucose are added into 1L of water, and the mixture is sterilized at 115 ℃ for 30 minutes.

(4) Method for detecting glucose and erythritol in fermentation process

And (3) diluting the fermentation liquor, filtering the fermentation liquor by using a 0.22-micron filter membrane, and detecting the contents of glucose and erythritol in the filtrate by adopting an HPLC (high performance liquid chromatography) mode. The chromatographic conditions were as follows: a detector, a difference detector; chromatographic column, calcium type strong cation exchange column; column temperature, 80 ℃; the mobile phase is redistilled; flow rate, 0.7 mL/min; sample volume, 10. mu.L.

The invention successfully realizes the high-efficiency screening of the erythritol high-yield strain with higher temperature tolerance from the arginine-deficient yarrowia lipolytica mutant strain, and has the characteristics of high screening speed, stable high-temperature resistance property, simplicity, high efficiency and the like.

The invention has the outstanding characteristics that:

(1) the invention provides a method for screening high-temperature resistant variant strains from wild yarrowia lipolytica variant strains by using arginine auxotrophic phenotype as an auxiliary marker, and the screening efficiency and the stability of high-temperature resistant characters of the variant strains are obviously improved compared with those of a common screening method.

(2) The temperature adaptation range of the high-temperature-resistant erythritol fermentation strain obtained by the method can be expanded to 34-35 ℃, and the cooling cost in the erythritol fermentation process can be obviously reduced. The conversion rate of the erythritol can reach more than 63%.

Drawings

FIG. 1 shows the growth conditions of yarrowia lipolytica strain CGMCC No.1431 and high temperature resistant variant GDMCC 61939 cultured at different temperatures.

FIG. 2 shows the comparison of OD values of yarrowia lipolytica starting strain CGMCC No.1431 (legend Y-2) and high temperature resistant variant GDMCC 61939 (legend A30) cultured at different temperatures.

FIG. 3 shows the variation of glucose mass percent, erythritol mass percent, pH value and OD value with fermentation time during the fermentation process of the high temperature resistant variant GDMCC 61939.

Figure 4 shows the change of glucose mass percent, erythritol mass percent, pH value and OD value along with the fermentation time in the fermentation process of the starting strain CGMCC No. 1431.

Detailed Description

The invention will now be further illustrated by the following examples, which will aid understanding of the invention, but are not limited thereto.

Example 1 acquisition of arginine auxotrophic Yeast variant

(1) Inoculating activated yarrowia lipolytica into YPD medium, shake-culturing for 24 hr, washing yeast cells with phosphate buffer solution of pH6.0 for 3 times, and adjusting OD with buffer solution⁶⁰⁰After reaching about 1.0, EMS was treated for 30 minutes at 2% (v/v), then centrifuged at 1000g for 10min to remove the mutagen from the supernatant to terminate the reaction, and the precipitated yeast cells were washed 3 times with a phosphate buffer solution of pH6.0 and then inoculated into 100 ml of YPD mediumShaking for 16 hr, diluting and spreading onto YPDA solid medium, and culturing at 30 deg.C for 3 days.

The YPD culture medium comprises the following components: 3.0g of yeast extract powder, 5.0g of peptone and 50g of anhydrous glucose, and water was added to the mixture to make 1.0 liter.

The YPDA culture medium formula is as follows: 3.0g of yeast extract powder, 5.0g of peptone, 50g of anhydrous glucose and 20g of agar powder, and water is added to the mixture until the volume is 1.0 liter.

EMS refers to ethyl methanesulfonate.

The phosphate buffer solution is a solution obtained by adjusting a 50mM phosphate solution to pH6.0 with a concentrated NaOH solution.

(2) And randomly selecting yeast single colonies growing on the YPDA plate in the step (1) by using a mechanical arm, respectively seeding the yeast single colonies on an YNB agar plate containing 5% (w/v) of glucose and a 5% (w/v) YNB agar plate added with 0.01% (w/v) of arginine, and culturing the yeast single colonies at 30 ℃ for 5 days. Yeast isolates growing only on the arginine-added plates were selected and seeded again on 5% (w/v) YNB agar plates and 5% (w/v) YNB agar plates supplemented with 0.02% (w/v) arginine to confirm the results. The isolated strain thus obtained is an arginine auxotrophic yeast variant. One of them, GDMCC 61939, was deposited at the institute of microbiology, academy of sciences of Guangdong province, Guangzhou city, Michelia Tokyo No. 100, 56.

The YNB refers to a yeast nitrogen base without amino acid, and comprises the following components:

composition (A)	Content (%)
		Biotin	0.000045
Calcium pantothenate	0.006
		Folic acid	0.00003
Inositol	0.03
		Nicotinic acid	0.006
P-aminobenzoic acid (PABA)	0.003
		Pyridoxine hydrochloride	0.006
Riboflavin	0.003
		Thiamine hydrochloride	0.006
Boric acid	0.0075
		Copper sulfate	0.075
Potassium iodide	0.0015
		Zinc sulfate	0.006
Potassium dihydrogen phosphate	15
		Magnesium sulfate	7.5
Sodium chloride	1.5
		Calcium chloride	1.5
Ammonium sulfate	75

Example 2 temperature Adaptation test of arginine deficient Yeast variants

(1) Inoculating arginine auxotrophic yeast variant GDMCC 61939 on YPDA plate, culturing at 30 deg.C, 33 deg.C, 36 deg.C, and 38 deg.C for 4d, wherein the growth conditions of different isolates are shown in figure 1;

(2) wild type yeast CGMCC No.1431 was spotted on YPDA plates, and cultured at 30 deg.C, 33 deg.C, 36 deg.C, and 38 deg.C for 4 days, with the growth conditions of different isolates shown in figure 1.

Example 3 growth curves of yarrowia lipolytica at different temperatures

(1) The growth curves of the starting strain and the thermostable variant R33 were determined using the same species as in example 1 at different culture temperatures, as shown in FIG. 2.

(2) The conditions used to perform the growth curve determination were:

inoculation amount: 5% (v/v);

the components of the culture medium: glucose 10% (w/v), yeast extract powder 1.0% (w/v), peptone 2.0% (w/v);

liquid loading of 500ml shake flask: 50 ml;

rotating speed of a shaking table: at 250 rpm.

Example 4 fermentation test of erythritol by arginine auxotrophic Yeast variant

2.5ml seed solution of the yarrowia lipolytica variant GDMCC 61939 cultured in YPD medium at 30 ℃ for 24h was inoculated into a 500ml triangular flask containing 25ml of sterilized fermentation medium, and shake flask fermentation was carried out on a shaker at 34 + -1 ℃ and 250rpm for 2d, wherein different glucose consumption and erythritol accumulation conditions were shown due to the addition of arginine at different concentrations, and erythritol conversion with the addition of arginine at 0.015% (w/v) showed the best performance, and the conversion rate reached 63% or more (see the following table).

The fermentation medium comprises the following components: YNB 0.67 (w/v), dextrose anhydrous 20% (w/v), arginine was added at various concentrations. (YNB was dissolved in a small amount of water and added to another sterilized component medium at room temperature through a sterilizing filter).

Experimental group	Arginine add-on% (w/v)	Erythritol% (w/v)	Conversion rate%
				1	0	2.2	23.6
2	0.005	5.4	45.4
				3	0.01	7.0	52.5
4	0.015	7.7	63.1
				5	0.02	7.5	60.2

Example 6 fermentation test of arginine auxotrophic Yeast variant

Inoculating 2.5ml seed liquid of yarrowia lipolytica variant GDMCC 61939 cultured for 24h in YPD medium at 30 ℃ into multiple groups of parallel 500ml triangular flasks filled with 25ml of sterilized fermentation medium, culturing for 60h in shaking table at 34 +/-1 ℃ and 280rpm, shaking for fermentation, and periodically extracting 3 bottles for measuring process parameters to obtain a fermentation process curve as shown in figure 3, wherein the glucose in the culture medium can be completely consumed within 60h, and the conversion rate of erythritol reaches 63.2%.

The fermentation medium comprises the following components: YNB 0.67 (w/v), anhydrous glucose 20% (w/v), and arginine 0.015% was added. (YNB was dissolved in a small amount of water and added to other sterilized components through a sterilizing filter at room temperature).

Example 7 erythritol fermentation test of wild-type arginine prototrophic yeast strains

Inoculating 2.5ml of wild yarrowia lipolytica CGMCC 1431 seed solution which is cultured for 24h by YPD medium at 30 ℃ into a plurality of groups of parallel 500ml triangular flasks which are filled with 25ml of sterilized fermentation medium, culturing and shaking the seeds for 60h on a shaking table at 34 +/-1 ℃ and 280rpm, and performing shaking fermentation, regularly extracting 3 flasks to determine process parameters to obtain a fermentation process curve as shown in figure 4, wherein the strain grows and ferments very slowly in the fermentation process at 34 ℃, and the generated erythritol amount is very little.

The fermentation medium comprises the following components: YNB 0.67 (w/v), anhydrous glucose 20% (w/v), no arginine was added. (YNB was dissolved in a small amount of water and added to another sterilized medium through a sterilizing filter at room temperature).

Claims

1. A breeding method for screening high-temperature resistant yarrowia lipolytica variant strains by using arginine auxotrophy as an indicator.

2. The breeding method as claimed in claim 1, characterized in that auxotrophic arginine strains are obtained by natural mutagenesis or physical or chemical mutagenesis and then yeast variant strains having growth at temperatures above 35 ℃ are selected from them.

3. The variant thermophilic lipolytic yarrowia lipolytica strain of claim 1, wherein the strain is deposited at the institute of microbiology, academy of sciences, Guangdong province under accession number GDMCC 61939.

4. The breeding method of claim 1 is applied to preparing erythritol through fermentation, and the specific method is to use the strain of claim 3 to produce erythritol through fermentation at 34-35 ℃ by using glucose as a carbon source, and further comprises the following steps:

(1) preparing a seed solution by using the strain of claim 3 and the following culture medium: 5-20% (w/v) of glucose, 0.03-2.5% (w/v) of yeast extract, 0.03-2.5% (w/v) of peptone, 0.02-0.5% (w/v) of monopotassium phosphate and 0.1-0.75% (w/v) of ammonium citrate;

(2) inoculating the seed liquid into a fermentation culture medium according to the proportion of 0.5-15%, wherein the culture medium comprises the following components: glucose 20-40% (w/v), YNB 0.1-1.5% (w/v), arginine 0-0.05% (w/v); carrying out shake flask fermentation for 60-120h at 33-36 ℃, and obtaining the erythritol fermentation liquid at the shake flask rotation speed of 200-400 rpm.